Recombinant Human ALPL (carrier-free)

Product Details

Source

Human ALPL, amino acid (Leu18 - Ser502) (Accession: NM_00478) was expressed in CHO cells. The C-terminus contains 6His-tag.

Molecular Mass

The 491 amino acid recombinant protein has a predicted molecular mass of approximately 54.2 kD. The DTT-reduced and non reduced protein migrates at approximately 70-75 kD by SDS-PAGE. The C-terminus contains 6His tag. The predicted N-terminal amino acid is Leu.

Purity

> 95%, as determined by Coomassie stained SDS-PAGE

Formulation

0.22 µm filtered protein solution is in 50 mM HEPES, 100 mM NaCl, 1 mM MgCl₂, 0.1 mM ZnCl₂, pH 7.0.

Endotoxin Level

Less than 0.1 EU per µg cytokine as determined by the LAL method

Concentration

25 µg size is bottled at 200 µg/mL. 100 µg size and larger sizes are lot-specific and bottled at the concentration indicated on the vial. To obtain lot-specific concentration and expiration, please enter the lot number in our Certificate of Analysis online tool.

Storage & Handling

Unopened vial can be stored at -20°C or -70°C for six months. For maximum results, quick spin vial prior to opening. Avoid repeated freeze/thaw cycles.

Activity

Human tissue-nonspecific alkaline phosphatase (ALPL) cleaves the fluorogenic peptide substrate, 4-methylumbelliferyl phosphate (4-MUP). The specific activity is > 180,000 pmol/min/µg in the presence of 0.0005 µg of recombinant human ALPL.

Application

Bioassay

Additional Product Notes

Human ALPL Enzymatic Assay

Human tissue-nonspecific alkaline phosphatase (ALPL) cleaves the fluorogenic peptide substrate, 4-Methylumbelliferyl phosphate (4-MUP). The specific activity is > 180,000 pmol/min/µg in the presence of 0.0005 µg of recombinant human ALPL.

• Assay Buffer: 50 mM Tris, 1 mM MgCl₂, pH 9.0
• hALPL Substrate: (Sigma, Cat. No. M8883), 25 mg/mL in diH₂O
• Recombinant human ALPL
• F16 Black Maxisorp Plate (Nunc, Cat. No 475515)
• Fluorescent plate reader (Model: SpectraMax M3 by Molecular Devices) or equivalent

Procedure

• Dilute the recombinant human ALPL to 0.01 ng/µL in assay buffer.
• Dilute the substrate to 50 µM in assay buffer.
• In a plate load 50 µL of the diluted human ALPL to the wells. Include a substrate blank of 50 µL assay buffer and 50 µL of diluted substrate.
• Start the reaction by adding 50 µL of 50 µM substrate to the wells.
• Read at excitation and emission wavelengths of 365 nm and 445 nm (top read) respectively in kinetic mode for 5 minutes.
• Calculate specific activity:

Specific Activity (pmol/min/µg) = Adjusted V_max* (RFU/min) x Conversion Factor** (pmol/RFU)
                                                                                           amount of enzyme (µg)

*Adjusted for substrate blank
**Derived using calibration standard 4-Methumbelliferone (Sigma, Cat. No. M1381)

Per Well:
Recombinant human ALPL: 0.0005 µg
Substrate: 25 µM

BioLegend carrier-free recombinant proteins provided in liquid format are shipped on blue-ice. Our comparison testing data indicates that when handled and stored as recommended, the liquid format has equal or better stability and shelf-life compared to commercially available lyophilized proteins after reconstitution. Our liquid proteins are verified in-house to maintain activity after shipping on blue ice and are backed by our 100% satisfaction guarantee. If you have any concerns, contact us at tech@biolegend.com.

Antigen Details

Structure

Homodimeric enzyme

Distribution

Osteoblast, bone marrow mesenchymal stem cells, liver, bone, kidney

Function

Bone and tooth mineralization, metabolism of nucleotides, sequentially hydrolyzes ATP, ADP and AMP

Interaction

Monoesters of phosphoric acid, inorganic pyrophosphate, phosphoethanolamine, and pyridoxal 5′-phosphate. Interacts with the ECM, collagen.

Bioactivity

Human ALPL activity is measured by its ability to cleave 4-Methylumbelliferyl phosphate (4-MUP). The accumulation of cleavage product is quantified by the increase in fluorescence at excitation and emission of 365 and 455 nm, respectively.

Cell Type

Embryonic Stem Cells, Mesenchymal Stem Cells, Neutrophils, Osteoblasts

Biology Area

Cell Biology, Immuno-Oncology, Stem Cells

Molecular Family

Enzymes and Regulators, Proteases

Antigen References

1. Weis MJ, et al. 1988. J Biol Chem. 263:12002.
2. Kim EE and Wyckoff HW. 1991. J Mol Biol. 218:449.
3. Mornet E. 2000. Human Mutation. 15:309.
4. Yang Y, et al. 2012. Front Immunol. 3:314.
5. Silvent J, et al. 2014. J Biol Chem. 289:24168.
6. Liu W, et al. 2018. Bone Res. 6:27.
7. Lou M, et al. 2019. Biochem Biophys Res Commun. 513:528.

Gene ID

249 View all products for this Gene ID

UniProt

View information about ALPL on UniProt.org

Documentation

• Technical Data Sheet (pdf)
• Certificate of Analysis
• Material Safety Data Sheet

Reviews

Related Pages & Pathways

Related FAQs

Why choose BioLegend recombinant proteins?

     • Each lot of product is quality-tested for bioactivity as indicated on the data sheet.
     • Greater than 95% Purity or higher, tested on every lot of product.
     • 100% Satisfaction Guarantee for quality performance, stability, and consistency.
     • Ready-to-use liquid format saves time and reduces challenges associated with reconstitution.
     • Bulk and customization available. Contact us.
     • Learn more about our Recombinant Proteins.

How does the activity of your recombinant proteins compare to competitors?

We quality control each and every lot of recombinant protein. Not only do we check its bioactivity, but we also compare it against other commercially available recombinant proteins. We make sure each recombinant protein’s activity is at least as good as or better than the competition’s. In order to provide you with the best possible product, we ensure that our testing process is rigorous and thorough. If you’re curious and eager to make the switch to BioLegend recombinants, contact your sales representative today!

What is the specific activity or ED50 of my recombinant protein?

The specific activity range of the protein is indicated on the product datasheets. Because the exact activity values on a per unit basis can largely fluctuate depending on a number of factors, including the nature of the assay, cell density, age of cells/passage number, culture media used, and end user technique, the specific activity is best defined as a range and we guarantee the specific activity of all our lots will be within the range indicated on the datasheet. Please note this only applies to recombinants labeled for use in bioassays. ELISA standard recombinant proteins are not recommended for bioassay usage as they are not tested for these applications.

Have your recombinants been tested for stability?

Our testing shows that the recombinant proteins are able to withstand room temperature for a week without losing activity. In addition the recombinant proteins were also found to withstand four cycles of freeze and thaw without losing activity.

Does specific activity of a recombinant protein vary between lots?

Specific activity will vary for each lot and for the type of experiment that is done to validate it, but all passed lots will have activity within the established ED50 range for the product and we guarantee that our products will have lot-to-lot consistency. Please conduct an experiment-specific validation to find the optimal ED50 for your system.

How do you convert activity as an ED50 in ng/ml to a specific activity in Units/mg?

Use formula Specific activity (Units/mg) = 10^6/ ED50 (ng/mL)

Certificate of Analysis